ES2298827T3 - PROCEDURE FOR OBTAINING N-CARBOXIANHYDRIDES FROM ALPHA-AMINO ACIDS PROTECTED WITH URETHANE. - Google Patents

Abstract

Procedure for obtaining urethane-protected alpha-amino acid N-carboxyanhydrides (UNCA), of formula I (See formula) in which R1 and R2, identical or different, together or independently represent one another, a hydrogen atom or a side chain of a natural or synthetic alpha-amino acid eventually carrying functional groups, when appropriate protected; R 3 represents a saturated or unsaturated, linear or branched C1-C10 alkyl radical or an aralkyl or alkylaryl radical having 7 to 14 carbon atoms, characterized in that an alpha-amino acid N-carboxyanhydride (NCA) of formula II (See formula) in that R1 and R2 have the same meaning as for formula I, is reacted with at least one equivalent, relative to the molar amount involved of NCA of formula II, of dicarbonate of formula III (See formula) in which R3 it has the same meaning as for formula I, in the presence of a catalytic amount of 1,4-diazabicyclo [2.2.2] octane, also called triethylenediamine (TEDA), in relation to the molar amount involved of NCA of formula II, in an organic solvent, with a melting point of less than about -20 ° C.

Description

Procedure for obtaining N-carboxyanhydrides of urethane protected alpha-amino acids.

The present invention relates to a new N-carboxyanhydride preparation process of urethane protected alpha-amino acids. He new procedure allows the synthesis of N-carboxyanhydrides of urethane protected alpha-amino acids from N-carboxyanhydrides of alpha-amino acids in the presence of an amount triethylenediamine catalyst, without adding amounts significant of a tertiary amine type base.

The N-carboxyanhydrides of alpha-amino acids (below designated under the NCA abbreviation), optionally protected, are agents of acylation frequently used for poly formation high molecular weight alpha-amino acids and for the dipeptide production. NCAs are very reactive compounds, that do not form, particularly through re-configuration, no secondary product not desired and whose only reaction byproduct is the dioxide of carbon. When the NCA reacts with an amine function free of an amino acid, carbon dioxide is released immediately and a dipeptide is formed, which in turn contains an amine function free. This amine will react with NCA to form a tripeptide, and so on. Thus, NCAs can be used in the formation of poly (alpha-amino acids) but cannot be used easily in sequential polypeptide synthesis, being difficult to control multiple side reactions of condensation such as oligomerization.

They have been described in the bibliography N-carboxyanhydrides of alpha-amino acids substituted by groups urethane, and are used in peptide syntheses. The substituent urethane provides a high degree of protection and minimizes the polymerization reactions during the coupling reaction. UCAs protected with urethane, abbreviated as UNCA to They then present all the advantages of unsubstituted NCAs without the inconvenience of the latter.

UNCA allows a controlled synthesis of polypeptides without requiring any pre-activation of carboxyl groups, and without no addition of additives such as the N-hydroxybenzotriazole. In this way, it is facilitated the purification of the peptides prepared in solution, due to that only the byproduct of the peptide synthesis reaction It is carbon dioxide.

UNCAs are also very useful as subjects premiums in the synthesis of hormones or medications anti-AIDS

The UNCA, which are in crystalline form in ambient temperature and pressure conditions are stable under standard laboratory handling conditions, storage, and under the conditions of peptide synthesis.

The two main routes of synthesis of the UNCA as of NCA they are the following:

1) UNCA can be synthesized by condensation of an alkyl or aralkyl chloroformate, such as  Fmoc-Cl (chloroformate 9-fluorophenylmethyloxycarbonyl) or chloroformate benzyl, with an NCA in the presence of at least a quantity stoichiometric of a tertiary amine. This tertiary amine, which is N-methylmorpholine usually allows to purify the hydrochloric acid released. Thus, the NCA is dissolved in an inert solvent, such as THF, and is cooled. 1.1 a were added 1.3 equivalents of alkyl or aralkyl chloroformate of one only once, and then at least 1.5 were slowly added equivalents of a tertiary amine, for example the N-methylmorpholine. The resulting suspension was left at rest for 1 to 2 hours, at a temperature between -25 and -5ºC. Then, hydrochloric acid was added slowly dissolved in dioxane until obtaining pH values of approximately 4-5. Amine Hydrochloride Tertiary thus formed was removed by filtration, and UNCA was concentrated and then crystallized.

All steps of the process are carried out under an inert atmosphere (N 2), and all solvents are dried in a 4 molecular molecular sieve before being used (William D. Fuller et al ., Urethane-protected-alpha-amino acid N-carboxyanhydrides and peptide synthesis, Biopolymers , 1996, 40, 183-205).

This route of synthesis is not appropriate for prepare certain N-carboxyanhydrides of protected alpha-amino acid, particularly those protected by a t-butoxycarbonyl radical, being t-butyl chloroformate very unstable for above -20 ° C or in the presence of tertiary amines.

2) UNCAs can also be synthesized by condensing a dialkyl dicarbonate with an NCA. This reaction releases a molecule of alcohol and a molecule of carbon dioxide. This synthesis must be performed imperatively in the presence of a high amount, at least an amount of 50% molar relative to the molar amount of NCA involved, of a tertiary amine such as N-methylmorpholine associated with a catalytic amount of DMAP ( 4-dimethylaminopyridine) or a pyridine (William D. Fuller et al ., Urethane-protected-alpha-amino acid N-carboxyan hydrides and peptide synthesis, Biopolymers , 1996, 40, 183-205). This synthetic route is particularly adapted for the synthesis of N-carboxyanhydrides of alpha-amino acids protected by a di-butoxycarbonyl radical using di-third-butyldicarbonate dicarbonate, see also Bull. Cham Soc. Japan, 69 (1996), 2309-2316.

Application WO 89/08643 describes Alpha N-carboxyanhydrides alpha-amino acids and N-thiocarboxyanhydrides of urethane-protected alpha-amino acids, from formula,

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one

in which R and R 'represent a hydrogen atom, an alkyl, cycloalkyl, cycloalkyl radical substituted by a substituted alkyl, aryl or aryl radical substituted, and at least one R or R 'group does not represent an atom of hydrogen; R '' represents an alkyl, aryl, alkyl radical substituted, or substituted aryl; Z represents an oxygen atom or of sulfur and n is 0, 1 or 2.

These compounds are prepared by reaction of NCA with a halogenoformate in an inert solvent, such as toluene, under anhydrous conditions, in the presence of a type base tertiary amine added in excess.

The existing synthesis procedures of UNCA are not satisfactory. Indeed, the best procedure described above herein, which uses a base of tertiary amine type in an amount at least equal to 50% molar with relation to the amount of NCA involved, provides some yields only of approximately 60%, with the condition that The solvents are dried on a 4 Å molecular sieve and that they are treated between -20 and -15ºC.

Surprisingly, it has been discovered that the use of triethylene diamine (TEDA) in a very low catalytic amount, less than 5% molar relative to the molar amount of NCA involved, without any addition of tertiary amine type base, It leads to good results.

Within the scope of the present invention, the abbreviation NCA designates the alpha-amino acid N-carboxyanhydride (s)
acid (s) and UNCA designates the N-carboxyanhydride (s) of urethane-protected alpha-amino acid (s).

In the sense of the present invention, by the term "catalytic amount" means an amount significantly lower and more specifically 50% lower at that required by stoichiometry.

The present invention relates to a procedure for obtaining N-carboxyanhydrides from  urethane protected alpha-amino acids (UNCA), from formula I

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2

in which R1 and R2, identical or different, together or independently represent one of the other, a hydrogen atom or a side chain of a natural or synthetic alpha-amino acid carrier possibly from functional groups, when appropriate protected; R 3 represents a linear alkyl radical in C 1 -C 10 saturated or unsaturated, linear or branched or an aralkyl or alkaryl radical of 7 to 14 atoms of carbon, characterized in that an N-carboxyanhydride of alpha-amino acid (NCA) of the formula II,

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3

in which R1 and R2 have the same meaning as for formula I, is reacted with at least one equivalent, in relation to the molar quantity involved in NCA of formula II, of dicarbonate of formula III

4

in which R3 has the same meaning that for formula I, in the presence of an amount catalytic of 1,4-diazabicyclo [2.2.2] octane, also called triethylene diamine (TEDA), in relation to molar amount involved of NCA of formula II, in a inert organic solvent with a melting point lower than approximately -20 ° C.

A natural alpha-amino acid or synthetic is a carrier amino acid in the first carbon of the chain of an amine function and a carboxylic acid function. The rest of the alpha-amino acid is called a chain side of the alpha-amino acid.

R1 and R2 are, when appropriate, protected with protective groups commonly used in the field of amino acids and peptides (Bodansky, principles of peptide synthesis, Springer-Verlag , 1984; Alpha amino acids N-carboxyanhydrides and related heterocycles, Hans R. Kricheldorf, Springer Verlag, 1987).

R1 and R2, identical or different, represent advantageously a hydrogen atom, an alkyl radical in C 1 -C 8 linear or branched, comprising possibly one or more usual substituent (s) in the field of amino acids and peptides. Substituents they are selected in particular from the group consisting of OH, SH, NH2, NHC (NH) NH2, CONH2, O-C 1 -C 6 alkyl, O-aryl in C 6 -C 10, S-C 1 -C 6 alkyl, COO-C 1 -C 6 alkyl, COO-aralkyl in C 5 -C 8, in Particularly the benzyl ester radical.

A group R1 or R2 can represent advantageously a cycloalkyl radical in C 5 -C 7, optionally substituted by one or more usual group (s) in the field of amino acids and peptides. Substituents are selected in particular among the group consisting of halogens, OH, O-C 1 -C 6 alkyl, C 6 -C 10 O-aryl, alkyl in C_ {1} -C_ {6}.

A group R1 or R2 can represent advantageously a phenyl, naphthyl, heteroaromatic radical of 5 or 6 members or indole eventually replaced with one or more usual group (s) in the field of amino acids and of the peptides. The substituents are selected in particular from among the group consisting of halogens, OH, O-C 1 -C 6 alkyl, C 6 -C 10 O-aryl, alkyl in C_ {1} -C_ {6}.

For obvious reasons of steric obstacles, R1 and R2 cannot simultaneously represent a cyclic radical. The term "cyclic radical" means said C 5 -C 7 cycloalkyl radical as well as said 5 or 6 membered phenyl, naphthyl, or heteroaromatic radical or indole

R1 and R2 can also form together a C 5 -C 7 cycloalkyl radical eventually replaced with one or more group (s) usual (s) in the field of amino acids and peptides Groups are selected in particular from the group constituted by halogens, OH, O-alkyl in C 1 -C 6, alkyl in C 1 -C 6, O-aryl in C_ {6} -C_ {10}.

In the case where R1 and R2 do not together form a C 5 -C 7 cycloalkyl radical, so minus one of the groups R1 and R2, as defined above, it advantageously represents a hydrogen atom.

In the compounds of formula II, the groups functional are advantageously protected with groups adequate protectors.

According to an advantageous variant of the invention, R3 represents the methyl radical, the ethyl radical, the radical third-butyl, the benzyl radical, the allyl radical or the 9-fluorenylmethyl radical. Indeed though there is a great variety of urethanes that can be used as groups protectors, only a few are widely used in synthesis peptide They can be cited in particular T-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxycarbonyl (Fmoc). By consequently, the N-carboxyanhydrides of alpha-amino acids protected by these substituents They are particularly interesting.

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The process according to the present invention it allows to avoid the use of a tertiary amine, in a very quantity raised from 50 to 200% molar relative to the molar amount of NCA involved. Tertiary amines used in the prior art, N-methylmorpholine and pyridine, pose Really numerous problems. Especially, its use implies work with a high dilution, the formation of parasitic products,  an additional stage of separation, difficult recycling and, in addition, It is very expensive.

The process according to the invention allows also significantly reduce reaction times that are a from now less than 24 hours and advantageously of the order of 1 to 4 hours, while for technique procedures Previously, reaction times varied from 30 hours to 5 days.

The process according to the invention allows obtain UNCA, whose purity measured by GPC (phase chromatography soda) is greater than 90%, advantageously greater than 95%, with a Very satisfactory yield, greater than 60% by mass.

According to an advantageous variant of the invention, the solvent is selected from the group consisting of ethers in C_ {4} -C_ {10} cyclic or linear, and by alkanes in C 1 -C 5 chlorinated. Advantageously, the Solvent is THF (tetrahydrofuran).

When the reaction solvent is THF, the amount of solvent introduced is generally comprised between 500 g and 2 kg of solvent per mole of NCA of formula II involved.

According to an advantageous variant of the procedure According to the present invention, the introduced amount of TEDA is between 0.1 and 5 molar% of TEDA, in relation to the quantity molar involved of NCA of formula II. Even more advantageously, the introduced amount of TEDA is between 0.2% and 1% molar, in relation to the molar amount involved of NCA of formula II.

The NCA of formula II is reacted advantageously with 1.1 to 1.5 equivalents of dicarbonate of formula III in the presence of TEDA, in particular in the presence of 0.2% to 1% molar of TEDA in relation to the molar amount involved of NCA of formula II.

The dicarbonate of formula III is introduced advantageously, in the form of a solution, in a part of the solvent, advantageously in 0.5 to 2.0 parts by weight in relation to to the total amount of weight involved in dicarbonate, so regulate in the reaction medium comprising the other part of the solvent, the NCA of formula II to be transform and TEDA. During the introduction of dicarbonate, the reaction medium temperature is maintained between -20 and 5 ° C, advantageously between -15 and 5 ° C, and even more advantageously between -10 and  0 ° C According to an advantageous variant of the invention, the reaction is Performs in an inert atmosphere.

The catalytic effect of TEDA allows a technique of gradual introduction of dicarbonate in the middle of reaction, which allows the control of exothermicity by stopping the introduction, thus avoiding any risk of a dangerous uncontrolled reaction.

The procedure allows working in a medium much more concentrated than, coupled with reaction times reduced, allows a substantial gain in productivity and It greatly limits the risks of polymerization.

At the end of the addition of the dicarbonate of formula III, the reaction medium is left advantageously low stirring, for at least 30 minutes at a temperature between -5 and 10ºC.

At the end of the addition of the dicarbonate of formula III, as soon as the reaction medium has been left optionally under stirring, the reaction medium is filtered, and then at least 80% is removed, advantageously approximately 90% of the solvent by evaporation under reduced pressure. Then, a non-solvent compound is added in a amount preferably equivalent to the amount of solvent of reaction removed by evaporation, in order to precipitate the UNCA of formula I, which is then recovered by filtration, when appropriate, after the removal of such groups adapted protectors.

According to a variant of the invention, the solvent is removed by evaporation under reduced pressure at a temperature between 15 and 30 ° C, advantageously at room temperature.

The non-solvent compound of UNCA is advantageously an alkane in C_ {5} -C_ {10} linear or branched, in particular heptane B.

According to a variant of the invention, the precipitate is then dried under vacuum at a temperature below 30 ° C

The following examples illustrate the present invention and are not limiting.

Example one

Preparation of Boc-Val-NCA

The abbreviation Val represents the alpha-amino acid valine. Val-NCA therefore represents the compound of the following formula:

5

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In a 1 liter double chamber reactor, equipped with a cryotmostat, a funnel, a circulation system  of nitrogen, a mechanical stirring and a thermometric probe, it introduce, after obtaining an inert environment with nitrogen and cooling to -5 ± 2 ° C:

- 204 g of THF,

- 37.5 g (0.26 mol) of Val-NCA,

- 0.15 g (1.3 mmol) of TEDA.

The reaction medium is stirred for ½ hour, and then a solution of 69 g (0.316) is slowly introduced moles) of (Boc) 2 O in 50 g of THF for 2 hours by means of a drip funnel, regulating the temperature at -5ºC ± 2 ° C A slight gas release appears.

The reaction medium is kept under stirring. for 1 hour after the end of the drip of (Boc) 2 O.

The reaction medium is filtered at 0 ° C in a inert pre-layer mounted with THF and the reactor is rinse as well as the inert pre-layer with 50 ml of THF.

The filtrates are put back in the reactor double chamber still under nitrogen and 300 ml of THF are distilled at a reaction medium temperature of 18 to 26 ° C with a pressure from 140 to 160 millibars.

300 ml (215 g) of heptane B are added to a temperature of 25 ° C. The UNCA product of valine precipitates. After, 300 ml of THF / heptane B mixture is distilled at 25 ° C with 900-100 millibars up to an average volume of reaction of approximately 100 ml. Then, 200 ml of heptane B at a temperature of approximately 25 ° C. The middle of reaction is allowed to cool to -10 ° C, temperature maintained for 1 hour. Filtration is performed on a filter # 3 sintered, in a nitrogen atmosphere at -10 ° C. The product is Dry in a vacuum oven at a temperature of 25 ± 5 ° C.

Thus, 51.6 g (yield 80.8%) are obtained. of a white powder with rotating power of 59.1º (C = 1, THF) whose Purity measured by GPC is 100%.

Example 2

Preparation of Boc-Ile-NCA

The abbreviation Ile represents the alpha-amino acid isoleucine. Ile-NCA therefore represents the compound of the following formula:

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6

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Proceed as in example 1 with:

- 200.9 g of THF,

- 40.0 g (0.255 mol) of Ile-NCA,

- 0.14 g (1.3 mmol) of TEDA,

and a solution of 66 g (0.302 mol) of (Boc) 2 O in 66 g of THF.

After filtration and drying, it is recover 51.5 g (yield 78.6%) of the expected product, with a melting point of 107.6 ° C and whose rotating power is 60.3 ° (C = 1, THF). The purity measured by GPC is 99.3%.

Example 3

Preparation of Boc-D-Phe-NCA

The abbreviation Phe represents the alpha-amino acid phenylalanine. Phe-NCA therefore represents the compound of the following formula:

7

Proceed as in Example 1, with the difference that the temperature is set at -17 ± 1 ° C with:

- 427 g of THF,

- 25 g (0.131 moles) of D-Phe-NCA,

- 0.07 g (0.65 mmol) of TEDA,

and a solution of 34.2 g (0.157 mol) of (Boc) 2 O in 21.5 g of THF.

After filtration and drying, it is recover 24.1 g (63% yield) of the product according to the expected 1 H NMR structure, and whose purity measured by GPC It is 95.2%.

Example 4

Preparation of N-ethoxycarbonyl-valine-N-carboxyanhydride (EtOC-Val-NCA)

In a 1 liter double chamber reactor equipped with a cryotmostat, a drip funnel, a system of nitrogen circulation, mechanical agitation and a probe thermometric, are introduced after obtaining an inert environment with nitrogen and cooling to -5 ± 2 ° C:

- 204 g of THF,

- 20.0 g (0.141 mol) of Val-NCA,

- 0.078 g (0.7 mmol) of TEDA.

The reaction medium is stirred for 30 minutes, and then slowly introduce 27.1 g (0.167 moles) of diethyl dicarbonate [(EtOC) 2 O] for 1 hour by means of a drip funnel, regulating the temperature at -5ºC ± 2 ° C A slight gas release appears.

The reaction medium is maintained with stirring. for 1 hour after the end of the drip of (EtOC) 2 O.

THF is concentrated at a medium temperature reaction from 18 to 26 ° C at a pressure of 140 to 160 millibars.

220 ml of heptane B are added at a temperature of 25 ° C, and poured into a large amount of ice water. He product precipitates. It is filtered on a filter # 3 sintered in a nitrogen atmosphere The product is dried in a vacuum oven at a temperature of 25 ± 5 ° C.

20.1 g (67% yield) of a white powder, whose 1 H NMR and 13 C NMR spectra are according to the expected structure.

Claims (15)

1. Procedure for obtaining N-carboxyanhydrides of urethane protected alpha-amino acids (UNCA), from formula I 8 wherein R1 and R2, identical or different, together or independently represent one another, a hydrogen atom or a side chain of a natural or synthetic alpha-amino acid eventually carrying functional groups, where appropriate protected; R 3 represents a saturated or unsaturated, linear or branched C 1 -C 10 alkyl radical or an aralkyl or alkylaryl radical having 7 to 14 carbon atoms, characterized in that an alpha-amino acid N-carboxyanhydride (NCA) of formula II 9 in which R1 and R2 have the same meaning that for formula I, it reacts with at least an equivalent, in relation to the molar amount involved of NCA of formula II, of dicarbonate of formula III 4 in which R3 has the same meaning that for formula I, in the presence of an amount catalytic of 1,4-diazabicyclo [2.2.2] octane, also called triethylene diamine (TEDA), in relation to molar amount involved of NCA of formula II, in a solvent organic, with a melting point less than about -20 ° C. 2. A method according to claim 1, characterized in that R1 and R2, identical or different, represent a hydrogen atom, a linear or branched C 1 -C 8 alkyl radical, optionally substituted, a C 1 -cycloalkyl radical 5} -C7, optionally substituted, a phenyl radical, a naphthyl, or a 5- or 6-membered heteroaromatic, or optionally substituted indole, with the proviso that R1 and R2 do not each simultaneously represent a cyclic radical; or R 1 and R 2 together form a C 5 -C 7 cycloalkyl radical, optionally substituted, the possible substituents being customary substituents in the field of amino acids and peptides. 3. Method according to claim 2, characterized in that the eventual substituents are selected from the group consisting of OH, SH, NH2, NHC (NH) NH2, CONH2, O-alkyl in C_ { 1} -C 6, O-aryl in C 1 -C 6, S-C 1 -C 6 alkyl, COO-C 1 -C 6 alkyl, COO-aralkyl at C 5 -C 8. 4. Method according to any of the preceding claims, characterized in that R3 represents the methyl radical, the ethyl radical, the third butyl radical, the benzyl radical, the allyl radical or the 9-fluorenylmethyl radical. 5. Method according to any of the preceding claims, characterized in that the solvent is selected from the group of C 4 -C 10 ethers and chlorinated C 1 -C 5 alkanes. 6. Method according to claim 5, characterized in that the solvent is THF. 7. Method according to any of the preceding claims, characterized in that the amount of TEDA is comprised between 0.1% and 5% molar, relative to the molar amount involved of NCA of formula II. Method according to claim 7, characterized in that the amount of TEDA is comprised between 0.2% and 1% molar, relative to the molar amount involved of NCA of formula II. 9. Method according to any of the preceding claims, characterized in that the NCA of formula II is reacted with 1.1 to 1.5 equivalents of dicarbonate of formula III, relative to the molar amount involved of NCA of formula II. Method according to any of the preceding claims, characterized in that the dicarbonate of formula III is introduced in the form of a solution in 0.5 and 2 parts by weight of the solvent, in relation to the amount by weight involved of dicarbonate, regularly in the medium. of reaction comprising the other part of the necessary solvent, the NCA of formula II to be transformed and the TEDA. Method according to claim 10, characterized in that the temperature of the reaction medium is maintained during the introduction of the dicarbonate between -20 and 5 ° C, advantageously between -10 and 0 ° C. 12. Method according to claim 10 or 11, characterized in that it then comprises the following steps:

i)

filter the reaction medium;

ii)

eliminate at least 80%, so advantageously about 90%, of the solvent by evaporation at reduced pressure; and then

iii)

add a compound non-solvent, in an amount equivalent to the amount of reaction solvent removed by evaporation, in order to precipitate the UNCA of formula I; Y

iv)

retrieve the UNCA of formula I by filtration, after removing said protecting groups if required.

13. Method according to claim 12, characterized in that the solvent is removed by evaporation under reduced pressure at a temperature between 15 and 30 ° C. 14. Method according to any of claims 12 or 13, characterized in that the non-solvent compound is a linear or branched C5-C10 alkane. 15. Process according to any of claims 12 to 14, characterized in that the precipitate is then dried under vacuum at a temperature below 30 ° C.